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1.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360810

RESUMO

Dendritic cells (DCs) are cells derived from the hematopoietic stem cells (HSCs) of the bone marrow and form a widely distributed cellular system throughout the body. They are the most efficient, potent, and professional antigen-presenting cells (APCs) of the immune system, inducing and dispersing a primary immune response by the activation of naïve T-cells, and playing an important role in the induction and maintenance of immune tolerance under homeostatic conditions. Thus, this review has elucidated the general aspects of DCs as well as the current dynamic perspectives and distribution of DCs in humans and in various species of animals that includes mouse, rat, birds, dog, cat, horse, cattle, sheep, pig, and non-human primates. Besides the role that DCs play in immune response, they also play a pathogenic role in many diseases, thus becoming a target in disease prevention and treatment. In addition, its roles in clinical immunology have also been addressed, which include its involvement in transplantation, autoimmune disease, viral infections, cancer, and as a vaccine target. Therefore, based on the current knowledge and understanding of the important roles they play, DCs can be used in the future as a powerful tool for manipulating the immune system.


Assuntos
Células Dendríticas/imunologia , Animais , Doenças Autoimunes/imunologia , Células Dendríticas/citologia , Humanos , Neoplasias/imunologia , Viroses/imunologia
2.
Nat Commun ; 12(1): 5029, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413303

RESUMO

Dendritic cells (DC) in the lung that induce Th17 differentiation remain incompletely understood, in part because conventional CD11b+ DCs (cDC2) are heterogeneous. Here, we report a population of cDCs that rapidly accumulates in lungs of mice following house dust extract inhalation. These cells are Ly-6C+, are developmentally and phenotypically similar to cDC2, and strongly promote Th17 differentiation ex vivo. Single cell RNA-sequencing (scRNA-Seq) of lung cDC2 indicates 5 distinct clusters. Pseudotime analysis of scRNA-Seq data and adoptive transfer experiments with purified cDC2 subpopulations suggest stepwise developmental progression of immature Ly-6C+Ly-6A/E+ cDC2 to mature Ly-6C-CD301b+ lung resident cDC2 lacking Ccr7 expression, which then further mature into CD200+ migratory cDC2 expressing Ccr7. Partially mature Ly-6C+Ly-6A/E-CD301b- cDC2, which express Il1b, promote Th17 differentiation. By contrast, CD200+ mature cDC2 strongly induce Th2, but not Th17, differentiation. Thus, Th17 and Th2 differentiation are promoted by lung cDC2 at distinct stages of maturation.


Assuntos
Asma/imunologia , Antígeno CD11b/imunologia , Células Dendríticas/imunologia , Pulmão/imunologia , Células Th17/imunologia , Células Th2/imunologia , Transferência Adotiva/métodos , Animais , Asma/metabolismo , Asma/patologia , Sequência de Bases , Antígeno CD11b/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Célula Única/métodos , Células Th17/citologia , Células Th2/citologia
3.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361114

RESUMO

Dendritic cells (DCs) can be divided by lineage into myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs). They both are present in mucosal tissues and regulate the immune response by secreting chemokines and cytokines. Inflammatory bowel diseases (IBDs) are characterized by a leaky intestinal barrier and the consequent translocation of bacterial lipopolysaccharide (LPS) to the basolateral side. This results in DCs activation, but the response of pDCs is still poorly characterized. In the present study, we compared mDCs and pDCs responses to LPS administration. We present a broad panel of DCs secreted factors, including cytokines, chemokines, and growth factors. Our recent studies demonstrated the anti-inflammatory effects of quercetin administration, but to date, there is no evidence about quercetin's effects on pDCs. The results of the present study demonstrate that pDCs can respond to LPS and that quercetin exposure modulates soluble factors release through the same molecular pathway used by mDCs (Slpi, Hmox1, and AP-1).


Assuntos
Antioxidantes/farmacologia , Síndrome da Liberação de Citocina/tratamento farmacológico , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Mieloides/efeitos dos fármacos , Quercetina/farmacologia , Animais , Antioxidantes/administração & dosagem , Células Cultivadas , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/metabolismo , Síndrome da Liberação de Citocina/patologia , Citocinas/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Camundongos , Células Mieloides/citologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Quercetina/administração & dosagem
4.
Methods Mol Biol ; 2350: 21-30, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331276

RESUMO

Confocal immunofluorescence microscopy is an advanced imaging technique routinely applied in the laboratory and clinics. Histological analyses are performed from tissue material. In general, a single fluorochrome per laser is employed, limiting simultaneous analysis to four antigens in one staining with a conventional 4-laser line microscope. Here, we describe a protocol for combining fluorochromes with the same excitation but different emission properties that allows for the analysis of six different antigens in confocal immunofluorescence microscopy with a conventional 4-laser line microscope. The proposed multiplexed method permits the identification and characterization of complex cell populations in rare tissue material.


Assuntos
Imunofluorescência/métodos , Lasers , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Corantes Fluorescentes , Humanos , Microscopia Confocal/métodos
5.
PLoS Comput Biol ; 17(5): e1008592, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34029312

RESUMO

During cell migration in confinement, the nucleus has to deform for a cell to pass through small constrictions. Such nuclear deformations require significant forces. A direct experimental measure of the deformation force field is extremely challenging. However, experimental images of nuclear shape are relatively easy to obtain. Therefore, here we present a method to calculate predictions of the deformation force field based purely on analysis of experimental images of nuclei before and after deformation. Such an inverse calculation is technically non-trivial and relies on a mechanical model for the nucleus. Here we compare two simple continuum elastic models of a cell nucleus undergoing deformation. In the first, we treat the nucleus as a homogeneous elastic solid and, in the second, as an elastic shell. For each of these models we calculate the force field required to produce the deformation given by experimental images of nuclei in dendritic cells migrating in microchannels with constrictions of controlled dimensions. These microfabricated channels provide a simplified confined environment mimicking that experienced by cells in tissues. Our calculations predict the forces felt by a deforming nucleus as a migrating cell encounters a constriction. Since a direct experimental measure of the deformation force field is very challenging and has not yet been achieved, our numerical approaches can make important predictions motivating further experiments, even though all the parameters are not yet available. We demonstrate the power of our method by showing how it predicts lateral forces corresponding to actin polymerisation around the nucleus, providing evidence for actin generated forces squeezing the sides of the nucleus as it enters a constriction. In addition, the algorithm we have developed could be adapted to analyse experimental images of deformation in other situations.


Assuntos
Movimento Celular/fisiologia , Núcleo Celular/fisiologia , Modelos Biológicos , Actinas/metabolismo , Algoritmos , Animais , Fenômenos Biomecânicos , Núcleo Celular/ultraestrutura , Forma Celular/fisiologia , Biologia Computacional , Simulação por Computador , Células Dendríticas/citologia , Células Dendríticas/fisiologia , Elasticidade/fisiologia , Camundongos , Microtecnologia , Imagem com Lapso de Tempo
6.
Chem Commun (Camb) ; 57(42): 5111-5126, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33977972

RESUMO

DC-SIGN, a lectin discovered two decades ago, plays a relevant role in innate immunity. Since its discovery, it has turned out to be a target for developing antiviral drugs based on carbohydrates due to its participation in the infection process of several pathogens. A plethora of carbohydrate multivalent systems using different scaffolds have been described to achieve this goal. Our group has made significant contributions to this field, which are revised herein.


Assuntos
Antracenos/química , Moléculas de Adesão Celular/química , Glicosídeos/química , Lectinas Tipo C/química , Receptores de Superfície Celular/química , Antracenos/farmacologia , Antivirais/química , Antivirais/farmacologia , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/farmacologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Vírus da Dengue/efeitos dos fármacos , Fulerenos/química , Lectinas Tipo C/metabolismo , Nanopartículas Metálicas/química , Conformação Molecular , Receptores de Superfície Celular/metabolismo , Zika virus/efeitos dos fármacos
7.
Mol Immunol ; 135: 165-169, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33901761

RESUMO

Antigen presentation is a key feature of classical dendritic cells (cDCs). Numerous studies have also reported in mouse that, upon inflammation, monocytes enter tissues and differentiate into monocyte-derived DCs (mo-DC) that have the ability to present antigens to T cells. However, a population of inflammatory cDCs sharing phenotypic features with mo-DC has been recently described, challenging the existence of in vivo-generated mo-DC. Here we review studies describing mouse mo-DC in the light of these findings, and evaluate the in vivo evidence for monocyte-derived antigen-presenting cells. We examine the strategies used to demonstrate the monocytic origin of these cells. Finally, we propose that mo-DC play a complementary role to cDCs, by presenting antigens to effector T cells locally in tissues.


Assuntos
Apresentação do Antígeno/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Monócitos/imunologia , Linfócitos T/imunologia , Animais , Antígenos/imunologia , Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , Camundongos , Monócitos/citologia
8.
PLoS One ; 16(4): e0249117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33836009

RESUMO

Macrophages and dendritic cells (DCs) are innate immune cells that play a key role in defense against pathogens. In vitro cultures of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) are well-established and valuable methods for immunological studies. Typically, commercially available recombinant GM-CSF is utilized to generate BMDCs and is also used to culture alveolar macrophages. We have generated a new HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF (~180 ng/ml) into complete media as an alternative to commercial GM-CSF. Differentiation of dendritic cells and expression of various markers were kinetically assessed using the GM-CSF HEK293T cell line, termed supGM-CSF and compared directly to purified commercial GMCSF. After 7-9 days of cell culture the supGM-CSF yielded twice as many viable cells compared to the commercial purified GM-CSF. In addition to differentiating BMDCs, the supGM-CSF can be utilized to culture functionally active alveolar macrophages. Collectively, our results show that supernatant from our GM-CSF HEK293T cell line supports the differentiation of mouse BMDCs or alveolar macrophage culturing, providing an economical alternative to purified GM-CSF.


Assuntos
Exocitose , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Células HEK293 , Humanos , Camundongos , Transgenes
9.
Life Sci ; 278: 119527, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33887349

RESUMO

Doxorubicin or Adriamycin, is one of the most widely used chemotherapeutic drug for treating a myriad of cancers. It induces cell death through multiple intracellular targets: reactive oxygen species generation, DNA-adduct formation, topoisomerase II inhibition, histone eviction, Ca2+ and iron hemostasis regulation, and ceramide overproduction. Moreover, doxorubicin-treated dying cells undergo cellular modifications that enable neighboring dendritic cell activation and enhanced presentation of tumor antigen. In addition, doxorubicin also aids in the immune-mediated clearance of tumor cells. However, the development of chemoresistance and cardiotoxicity side effect has undermined its widespread applicability. Several formulations of doxorubicin and co-treatments with inhibitors, miRNAs, natural compounds and other chemotherapeutic drugs have been essential in reducing its dosage-dependent toxicity and combating the development of resistance. Further, more advanced research into the molecular mechanism of chemoresistance development would be vital in improving the overall survivability of clinical patients and in preventing cancer relapse.


Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Antígenos de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Células Dendríticas/citologia , Sinergismo Farmacológico , Epigenômica , Hemostasia , Humanos , MicroRNAs/metabolismo , Recidiva Local de Neoplasia , Neoplasias/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais , Inibidores da Topoisomerase II/farmacologia
10.
Adv Healthc Mater ; 10(15): e2001899, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33928762

RESUMO

Vaccines are commonly administered subcutaneously or intramuscularly, and local immune cells, notably dendritic cells (DCs), play a significant role in transporting vaccine antigens and adjuvants to draining lymph nodes. Here, it is compared how soluble and biomaterial-mediated delivery of Toll-like receptor (TLR)-targeted adjuvants, monophosphoryl lipid A (MPLA, TLR4 ligand) and 5'-C-phosphate-G-3' DNA (CpG DNA, TLR9 ligand), modulate 3D chemotaxis of bone marrow-derived dendritic cells (BMDCs) toward lymphatic chemokine gradients. Within microfluidic devices containing 3D collagen-based matrices to mimic tissue conditions, soluble MPLA increases BMDC chemotaxis toward gradients of CCL19 and CCL21, while soluble CpG has no effect. Delivering CpG on poly(lactic-co-glycolic) acid microparticles (MPs) enhances BMDC chemotaxis compared to MPLA-encapsulated MPs, and when co-delivered, MPLA and CpG do not synergistically enhance BMDC migration. It is concluded that supplementing granulocyte-macrophage colony stimulating factor-derived BMDC culture with interleukin-4 is necessary to induce CCR7 expression and chemotaxis of BMDCs. Different cell subsets in BMDC culture upregulate CCR7 in response to soluble versus biomaterial-loaded MPLA and CpG, and CCR7 expression does not consistently correlate with functional migration. The results show both adjuvant type and delivery method influence chemotaxis of DCs, and these findings uncover new directions for the rational design of vaccine formulations.


Assuntos
Quimiotaxia , Células Dendríticas/citologia , Sistemas de Liberação de Medicamentos , Receptor 4 Toll-Like/agonistas , Receptor Toll-Like 9/agonistas , Animais , Medula Óssea , Feminino , Camundongos Endogâmicos C57BL
11.
J Mater Chem B ; 9(18): 3892-3899, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33928989

RESUMO

Immunotherapy is revolutionizing cancer treatment. Vaccination of antigenic peptides has been identified as a promising strategy for cancer immunotherapy while insufficient immune responses were stimulated due to low antigenicity. Moreover, immune checkpoint blockade therapy is still limited by a low objective response rate. In this work, cationic polymer-lipid hybrid nanovesicle (P/LNV)-based liposomes are designed to simultaneously deliver tumor vaccines composed of anionic antigen epitopes, toll-like receptor-9 agonist (TLR9), CpG (AE/CpG), and indoleamine-2,3-dioxygenase (IDO) inhibitor, 1-methyl-tryptophan (1-MT), to increase the immunogenicity of peptide antigens and meanwhile block the immune checkpoint. P/LNV liposomes efficiently enhanced the uptake of vaccines by dendritic cells (DCs) and improved the maturation of DCs indicated by the significantly increased percentage of CD86+MHCI+ DCs, resulting in a potent cytotoxic T-lymphocyte (CTL) response against B16-OVA tumor cells in vitro. Importantly, the combination immunotherapy showed significantly higher therapeutic efficiency towards melanoma tumors in mice, compared with an untreated or individual therapy modality. Mechanistically, the co-delivery system could elicit a strong cancer-specific T-cell response, as characterized by the remarkably increased infiltration of CD8+ T cells in the tumor and draining lymph nodes. Altogether, cationic liposomes delivered with tumor vaccines and IDO inhibitor provide a promising platform for cancer immunotherapy by provoking antitumor T-cell immunity and simultaneously reversing the immunosuppressive tumor microenvironment.


Assuntos
Ilhas de CpG , Epitopos/imunologia , Imunoterapia/métodos , Lipossomos/química , Melanoma Experimental/terapia , Triptofano/análogos & derivados , Animais , Ânions/química , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Cátions/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Epitopos/química , Lipídeos/química , Lipossomos/farmacologia , Melanoma Experimental/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas/química , Polímeros/química , Taxa de Sobrevida , Triptofano/química
12.
Int J Mol Sci ; 22(8)2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33920631

RESUMO

BACKGROUND: The adaptor protein Src homology 3 domain-binding protein 2 (SH3BP2) is widely expressed in immune cells. It controls intracellular signaling pathways. The present study was undertaken to investigate the role of SH3BP2 in a murine systemic lupus erythematosus model. METHODS: For the lupus model, we used Faslpr/lpr mice. Clinical and immunological phenotypes were compared between Faslpr/lpr and SH3BP2-deficient Faslpr/lpr mice. Splenomegaly and renal involvement were assessed. Lymphocyte subsets in the spleen were analyzed by flow cytometry. To examine the role of SH3BP2 in specific cells, B cell-specific SH3BP2-deficient lupus mice were analyzed; T cells and bone marrow-derived dendritic cells and macrophages were analyzed in vitro. RESULTS: SH3BP2 deficiency significantly reduced lupus-like phenotypes, presented as splenomegaly, renal involvement, elevated serum anti-dsDNA antibody, and increased splenic B220+CD4-CD8- T cells. Notably, SH3BP2 deficiency in B cells did not rescue the lupus-like phenotypes. Furthermore, SH3BP2 deficiency did not substantially affect the characteristics of T cells and macrophages in vitro. Interestingly, SH3BP2 deficiency suppressed the differentiation of dendritic cells in vitro and reduced the number of dendritic cells in the spleen of the lupus-prone mice. CONCLUSIONS: SH3BP2 deficiency ameliorated lupus-like manifestations. Modulating SH3BP2 expression could thus provide a novel therapeutic approach to autoimmune diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Lúpus Eritematoso Sistêmico/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Rim/citologia , Rim/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Linfócitos T/imunologia
13.
Int J Mol Sci ; 22(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921475

RESUMO

The preparation of dendritic cells (DCs) for adoptive cellular immunotherapy (ACI) requires the maturation of ex vivo-produced immature(i) DCs. This maturation ensures that the antigen presentation triggers an immune response towards the antigen-expressing cells. Although there is a large number of maturation agents capable of inducing strong DC maturation, there is still only a very limited number of these agents approved for use in the production of DCs for ACI. In seeking novel DC maturation agents, we used differentially activated human mast cell (MC) line LAD2 as a cellular adjuvant to elicit or modulate the maturation of ex vivo-produced monocyte-derived iDCs. We found that co-culture of iDCs with differentially activated LAD2 MCs in serum-containing media significantly modulated polyinosinic:polycytidylic acid (poly I:C)-elicited DC maturation as determined through the surface expression of the maturation markers CD80, CD83, CD86, and human leukocyte antigen(HLA)-DR. Once iDCs were generated in serum-free conditions, they became refractory to the maturation with poly I:C, and the LAD2 MC modulatory potential was minimized. However, the maturation-refractory phenotype of the serum-free generated iDCs was largely overcome by co-culture with thapsigargin-stimulated LAD2 MCs. Our data suggest that differentially stimulated mast cells could be novel and highly potent cellular adjuvants for the maturation of DCs for ACI.


Assuntos
Técnicas de Cultura de Células/métodos , Células Dendríticas/efeitos dos fármacos , Imunoterapia Adotiva , Mastócitos/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Apresentação do Antígeno/efeitos dos fármacos , Apresentação do Antígeno/imunologia , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Humanos , Mastócitos/citologia , Mastócitos/imunologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Tapsigargina/farmacologia
14.
Transfusion ; 61(6): 1845-1855, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33786883

RESUMO

BACKGROUND: One of the major challenges in cellular therapy is the establishment and validation of simple and fast production protocols meeting good manufacturing practice (GMP) requirements. Dendritic cells (DCs) are of particular therapeutic interest, due to their critical role in T cell response initiation and regulation. Conventional wisdom states that DC generation from monocytes is a time-consuming protocol, taking up to 7-9 days. STUDY DESIGN AND METHODS: This study systematically screened and validated numerous culture components and conditions to identify the minimal requirements, which can give rise to functional monocyte-derived antigen-presenting cells (MoAPCs) in less than 48 h (36 h MoAPC). A total of 36 h MoAPCs were evaluated in terms of surface marker expression, endocytic capability, and induction of antigen-specific T cell expansion via flow cytometry. RESULTS: Screening of media compositions, glucose concentrations, and surface marker kinetics, particularly DC-SIGN as a DC-specific marker, allowed the generation of DC-like APCs in 36 h (36 h MoAPCs). A total of 36 h MoAPCs displayed a similar phenotype to 48 h MoAPC and standard 7 d MoDCs in terms of HLA-DP,DQ,DR, CD83, and DC-SIGN expression, while CD1a was preferentially expressed in standard MoDCs. Functional evaluation revealed that 36 h MoAPCs displayed reduced endocytosis capabilities and IL-12p70 production. However, 36 h MoAPCs were able to induce T cell expansion both in an allogenic and antigen-specific setting. CONCLUSION: Our results indicate that mature 36 h MoAPCs possess DC-like capabilities by inducing antigen-specific T cell responses. This study has important implications for the generation of DC-based cellular therapies, allowing a more cost and time-efficient generation of APCs.


Assuntos
Células Apresentadoras de Antígenos/citologia , Células Dendríticas/citologia , Monócitos/citologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Meios de Cultura/metabolismo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Monócitos/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo
15.
J Hematol Oncol ; 14(1): 43, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731208

RESUMO

BACKGROUND: Transgelin-2 is a 22 kDa actin-binding protein that has been proposed to act as an oncogenic factor, capable of contributing to tumorigenesis in a wide range of human malignancies. However, little is known whether this tiny protein also plays an important role in immunity, thereby keeping body from the cancer development and metastasis. Here, we investigated the functions of transgelin-2 in dendritic cell (DC) immunity. Further, we investigated whether the non-viral transduction of cell-permeable transgelin-2 peptide potentially enhance DC-based cancer immunotherapy. METHODS: To understand the functions of transgelin-2 in DCs, we utilized bone marrow-derived DCs (BMDCs) purified from transgelin-2 knockout (Tagln2-/-) mice. To observe the dynamic cellular mechanism of transgelin-2, we utilized confocal microscopy and flow cytometry. To monitor DC migration and cognate T-DC interaction in vivo, we used intravital two-photon microscopy. For the solid and metastasis tumor models, OVA+ B16F10 melanoma were inoculated into the C57BL/6 mice via intravenously (i.v.) and subcutaneously (s.c.), respectively. OTI TCR T cells were used for the adoptive transfer experiments. Cell-permeable, de-ubiquitinated recombinant transgelin-2 was purified from Escherichia coli and applied for DC-based adoptive immunotherapy. RESULTS: We found that transgelin-2 is remarkably expressed in BMDCs during maturation and lipopolysaccharide activation, suggesting that this protein plays a role in DC-based immunity. Although Tagln2-/- BMDCs exhibited no changes in maturation, they showed significant defects in their abilities to home to draining lymph nodes (LNs) and prime T cells to produce antigen-specific T cell clones, and these changes were associated with a failure to suppress tumor growth and metastasis of OVA+ B16F10 melanoma cells in mice. Tagln2-/- BMDCs had defects in filopodia-like membrane protrusion and podosome formation due to the attenuation of the signals that modulate actin remodeling in vitro and formed short, unstable contacts with cognate CD4+ T cells in vivo. Strikingly, non-viral transduction of cell-permeable, de-ubiquitinated recombinant transgelin-2 potentiated DC functions to suppress tumor growth and metastasis. CONCLUSION: This work demonstrates that transgelin-2 is an essential protein for both cancer and immunity. Therefore, transgelin-2 can act as a double-edged sword depending on how we apply this protein to cancer therapy. Engineering and clinical application of this protein may unveil a new era in DC-based cancer immunotherapy. Our findings indicate that cell-permeable transgelin-2 have a potential clinical value as a cancer immunotherapy based on DCs.


Assuntos
Transferência Adotiva , Células Dendríticas/imunologia , Melanoma Experimental/terapia , Proteínas dos Microfilamentos/imunologia , Proteínas Musculares/imunologia , Animais , Movimento Celular , Células Cultivadas , Células Dendríticas/citologia , Feminino , Imunidade , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética
16.
FASEB J ; 35(5): e21418, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33774873

RESUMO

Constitutively expressed by innate immune cells, the cytokine macrophage migration inhibitory factor (MIF) initiates host immune responses and drives pathogenic responses in infectious, inflammatory, and autoimmune diseases. Dendritic cells (DCs) express high levels of MIF, but the role of MIF in DC function remains poorly characterized. As migration is critical for DC immune surveillance, we investigated whether MIF promoted the migration of DCs. In classical transwell experiments, MIF-/- bone marrow-derived DCs (BMDCs) or MIF+/+ BMDCs treated with ISO-1, an inhibitor of MIF, showed markedly reduced spontaneous migration and chemotaxis. CD74-/- BMDCs that are deficient in the ligand-binding component of the cognate MIF receptor exhibited a migration defect similar to that of MIF-/- BMDCs. Adoptive transfer experiments of LPS-matured MIF+/+ and MIF-/- and of CD74+/+ and CD74-/- BMDCs injected into the hind footpads of homologous or heterologous mice showed that the autocrine and paracrine MIF activity acting via CD74 contributed to the recruitment of DCs to the draining lymph nodes. Mechanistically, MIF activated the Src/PI3K signaling pathway and myosin II complexes, which were required for the migration of BMDCs. Altogether, these data show that the cytokine MIF exerts chemokine-like activity for DC motility and trafficking.


Assuntos
Antígenos de Diferenciação de Linfócitos B/fisiologia , Quimiotaxia , Células Dendríticas/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Miosina Tipo II/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Quinases da Família src/metabolismo , Animais , Células Cultivadas , Quimiocinas/metabolismo , Células Dendríticas/citologia , Imunidade , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosina Tipo II/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais , Quinases da Família src/genética
17.
Front Immunol ; 12: 580594, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33767691

RESUMO

The zoonotic intracellular bacterium Chlamydia psittaci causes life-threatening pneumonia in humans. During mouse lung infection, complement factor C3 and the anaphylatoxin C3a augment protection against C. psittaci by a so far unknown mechanism. To clarify how complement contributes to the early, innate and the late, specific immune response and resulting protection, this study addresses the amount of C3, the timing when its presence is required as well as the anaphylatoxin receptor(s) mediating its effects and the complement-dependent migration of dendritic cells. Challenge experiments with C. psittaci on various complement KO mice were combined with transient decomplementation by pharmacological treatment, as well as the analysis of in vivo dendritic cells migration. Our findings reveal that a plasma concentration of C3 close to wildtype levels was required to achieve full protection. The diminished levels of C3 of heterozygote C3+/- mice permitted already relative effective protection and improved survival as compared to C3-/- mice, but overall recovery of these animals was delayed. Complement was in particular required during the first days of infection. However, additionally, it seems to support protection at later stages. Migration of CD103+ dendritic cells from the infected lung to the draining lymph node-as prerequisite of antigen presentation-depended on C3 and C3aR and/or C5aR. Our results provide unique mechanistic insight in various aspects of complement-dependent immune responses under almost identical, rather physiological experimental conditions. Our study contributes to an improved understanding of the role of complement, and C3a in particular, in infections by intracellular bacteria.


Assuntos
Movimento Celular/imunologia , Infecções por Chlamydiaceae/imunologia , Chlamydophila psittaci/imunologia , Complemento C3a/imunologia , Células Dendríticas/imunologia , Pulmão/imunologia , Anafilatoxinas/imunologia , Anafilatoxinas/metabolismo , Animais , Linhagem Celular , Infecções por Chlamydiaceae/metabolismo , Infecções por Chlamydiaceae/microbiologia , Chlamydophila psittaci/fisiologia , Ativação do Complemento/imunologia , Complemento C3a/genética , Complemento C3a/metabolismo , Células Dendríticas/citologia , Células Dendríticas/microbiologia , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Complemento/genética , Receptores de Complemento/imunologia , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia , Análise de Sobrevida
18.
Cell ; 184(7): 1836-1857.e22, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33713619

RESUMO

COVID-19 exhibits extensive patient-to-patient heterogeneity. To link immune response variation to disease severity and outcome over time, we longitudinally assessed circulating proteins as well as 188 surface protein markers, transcriptome, and T cell receptor sequence simultaneously in single peripheral immune cells from COVID-19 patients. Conditional-independence network analysis revealed primary correlates of disease severity, including gene expression signatures of apoptosis in plasmacytoid dendritic cells and attenuated inflammation but increased fatty acid metabolism in CD56dimCD16hi NK cells linked positively to circulating interleukin (IL)-15. CD8+ T cell activation was apparent without signs of exhaustion. Although cellular inflammation was depressed in severe patients early after hospitalization, it became elevated by days 17-23 post symptom onset, suggestive of a late wave of inflammatory responses. Furthermore, circulating protein trajectories at this time were divergent between and predictive of recovery versus fatal outcomes. Our findings stress the importance of timing in the analysis, clinical monitoring, and therapeutic intervention of COVID-19.


Assuntos
COVID-19/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Expressão Gênica/imunologia , Células Matadoras Naturais/metabolismo , Índice de Gravidade de Doença , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , COVID-19/mortalidade , Estudos de Casos e Controles , Células Dendríticas/citologia , Feminino , Humanos , Células Matadoras Naturais/citologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Transcriptoma/imunologia , Adulto Jovem
19.
Front Immunol ; 12: 602122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33746947

RESUMO

Clever-1 also known as Stabilin-1 and FEEL-1 is a scavenger molecule expressed on a subpopulation of anti-inflammatory macrophages and lymphatic endothelial cells (LECs). However, its role in regulating dendritic cell (DC) trafficking and subsequent effects on immunity have remained unexplored. In this study, we demonstrate that DC trafficking from the skin into the draining lymph nodes is compromised in the absence of Clever-1. By adoptive transfer approaches we further show that the poor trafficking is due to the impaired entrance of DCs into afferent lymphatics. Despite this, injections of ovalbumin-loaded DCs into the footpads induced a stronger proliferative response of OT II T cells in the draining lymph nodes. This could be explained by the increased MHC II expression on DCs and a less tolerogenic phenotype of LECs in lymph nodes of Clever-1 knockout mice. Thus, although fewer DCs reach the nodes, they are more active in creating antigen-specific immune responses. This suggests that the DCs migrating to the draining lymph node within Clever-1 positive lymphatics experience immunosuppressive interactions with LECs. In conclusion, besides being a trafficking molecule on lymphatic vasculature Clever-1 is immunosuppressive towards migrating DCs and thus, regulates the magnitude of immune responses created by incoming DCs in the draining lymph nodes.


Assuntos
Moléculas de Adesão Celular Neuronais/deficiência , Células Dendríticas/imunologia , Células Endoteliais/imunologia , Deleção de Genes , Migração Transendotelial e Transepitelial/imunologia , Animais , Moléculas de Adesão Celular Neuronais/imunologia , Células Dendríticas/citologia , Células Endoteliais/citologia , Camundongos , Camundongos Knockout , Migração Transendotelial e Transepitelial/genética
20.
Nature ; 590(7846): 457-462, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33568812

RESUMO

In contrast to nearly all other tissues, the anatomy of cell differentiation in the bone marrow remains unknown. This is owing to a lack of strategies for examining myelopoiesis-the differentiation of myeloid progenitors into a large variety of innate immune cells-in situ in the bone marrow. Such strategies are required to understand differentiation and lineage-commitment decisions, and to define how spatial organizing cues inform tissue function. Here we develop approaches for imaging myelopoiesis in mice, and generate atlases showing the differentiation of granulocytes, monocytes and dendritic cells. The generation of granulocytes and dendritic cells-monocytes localizes to different blood-vessel structures known as sinusoids, and displays lineage-specific spatial and clonal architectures. Acute systemic infection with Listeria monocytogenes induces lineage-specific progenitor clusters to undergo increased self-renewal of progenitors, but the different lineages remain spatially separated. Monocyte-dendritic cell progenitors (MDPs) map with nonclassical monocytes and conventional dendritic cells; these localize to a subset of blood vessels expressing a major regulator of myelopoiesis, colony-stimulating factor 1 (CSF1, also known as M-CSF)1. Specific deletion of Csf1 in endothelium disrupts the architecture around MDPs and their localization to sinusoids. Subsequently, there are fewer MDPs and their ability to differentiate is reduced, leading to a loss of nonclassical monocytes and dendritic cells during both homeostasis and infection. These data indicate that local cues produced by distinct blood vessels are responsible for the spatial organization of definitive blood cell differentiation.


Assuntos
Rastreamento de Células/métodos , Células Mieloides/citologia , Mielopoese , Coloração e Rotulagem/métodos , Animais , Atlas como Assunto , Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Linhagem da Célula , Autorrenovação Celular , Células Dendríticas/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Granulócitos/citologia , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Fator Estimulador de Colônias de Macrófagos/deficiência , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Camundongos , Monócitos/citologia , Células Mieloides/metabolismo
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